High amylose wheat starch and wheat containing the same

ABSTRACT

Wheat starch of the present invention is obtained from endosperm of a seed of wheat which is modified to lack starch granule protein-1 (SGP-1). The wheat starch has an apparent amylose content of about 35% or more. Wheat flour of the present invention is obtained from endosperm of a seed of wheat which is modified to lack SGP-1. Wheat of the present invention is modified to lack SGP-1. The wheat flour and the wheat comprise wheat starch which has an apparent amylose content of about 35% or more.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.09/741,227, filed Dec. 19, 2000, now abandoned which is a divisional ofU.S. application Ser. No. 09/325,819, filed Jun. 4, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to wheat starch having novel propertiesand, more particularly, to wheat starch having a high apparent amylosecontent.

2. Description of the Related Art

Starch is the major component of the endosperm of a cereal seed such aswheat. Wheat starch components can be either amylose or amylopectin. Theamylose content of wheat starch is 0% for waxy wheat cultivars and about22-30% (about 29% on average) for normal (nor-waxy) wheat cultivars.

Some maize cultivars yield corn starch in which the amylose content isas high as about 60-70%. Cornstarch with high amylose content providesvarious industrial applications, such as an adhesive for cardboard, aconverging agent for glass fiber and an edible film, as well as foodapplications such as ricemeal which is used when making rice cake.

Among various rice varieties, Indica rice grain has a higher amylosecontent than Japonica rice grain. Rice grains having a high amylosecontent can be suitably used for pilaf and rice vermicelli.

Wheat starch with a high amylose content and wheat flour containing suchwheat starch are expected to provide new industrial and foodapplications. Therefore, attempts have been made to produce wheat starchwith increased amylose content using crossbreeding and geneticengineering approaches. However, to the extent the present inventor isaware of, no satisfactory results have been obtained.

Amylose is an α(1,4)-linked glucose polymer which is essentially alinear chain without branching. Amylopectin is a branched glucosepolymer in which branch chains are linked to the main chain of α(1,4)-linked polymer by α(1,6)-linkages. The linear glucose polymers aresynthesized by the action of starch synthases which produces(1,4)-linkages. The (1,6)-linkages of amylopectin are produced by theaction of branching enzymes.

Studies in pea, maize, and wheat (Denyer et al., Plant J. 4:191-198,1993; Echt and Schwartz, Genetics 99:275-284, 1981; Mu et al., Plant J.6:151-159, 1994: and Denyer et al., Planta 196:256-265, 1995) have shownthat some enzymes for starch synthesis are tightly bound to starchgranules from seed endosperms of maize and wheat and pea embryo.

The detailed mechanism for the binding of these enzymes to starchgranules has been unknown. However, it is believed that in wheat, atleast four kinds of proteins, i.e., waxy protein and three starchgranule proteins (SGP-1, SGP-2, SGP-3), are tightly bound to starchgranules and are responsible for starch synthesis. Waxy protein, i.e.,granule-bound starch synthase I (GBSS I) responsible for amylosesynthesis, is the product of the waxy gene (Ainsworth et al., Plant MolBiol. 22:67-82, 1993). SGP-1, -2 and -3 (Yamamori and Endo, Theor Appl.Genet. 93:275-281, 1996) correspond to starch granule-bound isozymes ofabout 100-105 kDa, about 90 kDa and about 77 kDa , respectively,reported by Denyer et al. (Planta, supra). Immunoblotting, amino acidsequencing and detection of starch synthase or branching activities(Denyer et al., Planta, supra: Rahman et al., Aust. J. Plant Physiol.22:793-803, 1995; Takaoka et al., J. Agric. Food Chem. 45:2929-2934,1997) suggest that SGP-2 is a homolog of maize branching enzyme IIb(Fisher et al., Plant Physiol. 102:1045-1046, 1993) and that SGP-3 is ahomolog of maize starch synthase I (Knight et al., Plant J. 14:613-622,1998).

Immunoblotting studies on about 100-105 kDa protein (SGP-1) did notdetect the protein in the soluble fraction. Thus, SGP-1 is exclusivelybound to starch granules (Denyer et al., Planta, supra; Rahman et al.,supra) This protein is presumed to be a starch synthase from the studiesof antiserum recognition, enzymatic activity detected and homology inamino acid sequences (Denyer et al., Planta, supra; Takaoka et al.,supra). However, information regarding the physiological function ofSGP-1 in vivo has been limited. For maize, it has been reported that anapparent amylose content is increased in a mutant of dull 1 gene whichis presumed to code for starch synthase II (Gao et al., The Plant Cell10:399-412, 1998). However, there is no substantial amino acid sequencehomology between the protein coded by dull 1 (Gao at al., supra) and theprotein SGP-1 of wheat (Takaoka et al., supra). Further, the proteincoded by dull 1 is present in the soluble fraction. Thus, the starchsynthase encoded by dull 1is significantly different from SGP-1.

A hexaploid wheat has three isozymes of SGP-1, i.e., SGP-A1, SGP-B1 andSGP-D1. The gene coding for SGP-A1, Sgp-A1, is located on chromosome arm7A, Sgp-B1 on 7B, and Sgp-D1 on 7D (Denyer et al., Planta, supra). UsingSDS-polyacrylamide gel electrophoresis (SDS-PAGE), it has been foundthat a few wheat cultivars lacked either SGP-A1,-B1 or -D1, but no wheatcultivars lacked two or more SGP-1s (Yamamori and Endo, supra).

SUMMARY OF THE INVENTION

According to one aspect of this invention, there is provided wheatstarch obtained from endosperm of a seed of wheat which is modified tolack starch granule protein-1 (SGP-1). The wheat starch has an apparentamylose content of about 35% or more.

According to another aspect of this invention, there is provided wheatflour obtained from endosperm of a seed of wheat which is modified tolack SGP-1. The wheat flour includes wheat starch which has an apparentamylose content of about 35% or more.

According to still another aspect of this invention, there is providedwheat which is modified to lack SGP-1. The wheat includes wheat starchwhich has an apparent amylose content of about 35% or more.

In one embodiment, the apparent amylose content may be from about 30% toabout 45%, preferably from about 37% to about 40%.

In one embodiment, the wheat may be a hexaploid wheat which lacksSGP-A1, SGP-B1 and SGP-D1 The hexaploid wheat may be obtained bycrossing a first wheat lacking a first protein selected from the groupconsisting of SGP-A1, SGP-B1 and SGP-D1, with a second wheat lacking asecond protein which differs from the first protein and is selected fromthe group consisting of SGP-A1, SGP-B1 and SGP-D1, followed by furthercrossing the cross of the first wheat and the second wheat with a thirdwheat lacking a third protein which differs from the first and secondproteins and is selected from the group consisting of SGP-A1, SGP-B1 andSGP-D1. The hexaploid wheat may be obtained by crossing (i) Chousen 30or Chousen 57, (ii) Turkey 116, and (iii) Kanto 79 in an arbitraryorder.

Thus, the invention described herein makes possible the advantage ofproviding wheat starch having a high apparent amylose content.

This and other advantages of the present invention will become apparentto those skilled in the art upon reading and understanding the followingdetailed description with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a gel developing an electrophoresis pattern ofwheat lacking one or more SGP-1s. Lane 1 shows Chinese Spring as acontrol; lane 2 shows Turkey 116: lane 3 shows Kanto 79; lane 4 showsChousen 57: and lane 5 shows SGP-1 null wheat.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention will be described in greater detail.

1. Definitions

“SGP-1” is one of several kinds of proteins referred to as starchgranule proteins, or “SGPs”, which are not washed off, but remain boundto, starch granules of wheat seed endosperms during a process forwashing the starch granules with a buffer containing a surfactant,sodium dodecyl sulfate (SDS). Recent reports have shown that SGP-1 is astarch synthase. Hexaploid wheat having genome organization of AABBDDhas three isozymes of SGP-1, i.e., SGP-A1, -B1 and -D1. Tetraploid wheathaving genome organization of AABB has two isozymes of SGP-1, i.e.,SGP-A1 and -B1. These isozymes of SGP-1 can be detected and identifiedby SDS-gel electrophoreses as three distinguished protein bands.Specifically, SGP-A1, -B1 and -D1 are detected by SDS-gelelectrophoresis as bands of about 115 kDa, about 100 kDa and about 108kDa, respectively (Yamamori and Endo, supra).

Herein, the terms “SGP-1”, “SGP-A1”, “SGP-B1” and “SGP-D1” are used todenote proteins, while “Sgp-1”, “Sgp-A1”, “Sgp-B1” and “Sgp-D1” are usedto denote genes coding for the proteins, SGP-1, SGP-A1, SGP-B1 andSGP-D1, respectively.

The phrase “lacking” SGP-1 as used herein means that any SGP-1 proteinis not expressed at a level detectable in SDS-gel electrophoresis. Morespecifically, it means that the band of the protein of interest issubstantially undetectable by silver staining which is a sensitivemethod for protein detection.

The term “apparent amylose content” as used herein refers to an amylosecontent as measured by calorimetric measurement based on iodinecoloration using an auto-analyzer or by amperometric titration based oniodine affinity. In the context of the present invention, when it isstated that wheat has an apparent amylose content of “about 35% ormore”, for example, means that the wheat has an amylose content of about35% or more as measured under conditions that are substantially the sameas those used in either the colorimetric measurement as described insection (1) of Example 4below or the amperometric titration as describedin section (2) of Example 4 below, or both.

The wheat starch herein disclosed can also be characterized by maximumabsorbance (λ_(max)) and absorbance at 680 nm (blue value). As measuredunder substantially the same conditions as those of Example 3 below, thewheat starch may have λ_(max) of from about 600 nm to about 620 nm, andblue value of from about 0.45 to about 0.55.

“Wheat” refers to a plant belonging to the genus Triticum. Wheatincludes “hexaploid wheat” which has genome organization of AABBDD,comprised of 42 chromosomes, and “tetraploid wheat” which has genomeorganization of AABB, comprised of 28 chromosomes. Hexaploid wheatincludes T. aestivum, T. spelta, T. macha, T. compactum, T.sphaerococcum, T. vavilovii, and interspecies cross thereof. Tetraploidwheat includes T. durum, T. dicoccoides, T. dicoccum, T. polonicum, andinterspecies cross thereof. A wheat cultivar for use in the presentinvention may belong to any of the above-listed species, and preferablya hexaploid wheat, and more preferably T. aestivum.

“Modified” wheat as used herein refers to wheat which has beenartificially manipulated to lack SGP-1, and it is intended to excludenaturally-occurring wheat The artificial manipulation of wheat istypically, but not limited to, cross breeding. It may be any otherappropriate manipulation, including mutagenesis and geneticrecombination.

“Kanto 79/Turkey 116” as used herein refers to a cross obtained bypollinating Kanto 79 with pollen of Turkey 116. “(Kanto 79/Turkey116)F₂//Chousen 57” as used herein refers to a cross obtained by firstpollinating Kanto 79 with pollen of Turkey 116 to obtain a plant (F₁),self-pollinating the plant (F₁) to obtain a new progeny plant (F₂); andthen pollinating the progeny plant (F₂) with pollen of Chousen 57.

2. Production of Wheat Lacking SGP-1 (SGP-1 Null Wheat)

As a method for completely eliminating all SGP-1 proteins from hexaploidwheat, the present inventors have developed a novel method as will bedescribed below It is noted that as a pollen parent for the crossingprocess which will be described below, either of the parent wheatcultivars can be used because SGP-1 is coded by a chromosomal gene.

First, a wheat cultivar lacking only SGP-D1 (Sgp-D1 null), for example,is crossed with another wheat cultivar lacking only SGP-B1 (Sgp-B1 null)so as to obtain F₁ seeds. Since a wheat cultivar is generally ahomozygote, the obtained F₁ seeds will be heterozygous for both Sgp-D1and Sgp-B1, whereby both SGP-D1 and SGP-B1 will be detected in the F₁seed endosperms. When F₁ plants which have grown from the F₁ seeds areself-pollinated, F₂ seeds will segregate with regard to each of Sgp-D1and Sgp-B1 alleles at the probability of one out of four (¼). That is,one out of four F₂ seeds will be null as to the Sgp-B1 gene, andindependently, one out of four F₂ seeds will be null as to the Sgp-D1gene. Thus, from the entire F₂ seed population, an F₂ seed being null asto both Sgp-D1 and Sgp-B1 is obtained by the probability of one out ofsixteen ({fraction (1/16)}; i.e., ¼ multiplied by ¼), theoretically.

Starches are purified from distal halves of the obtained F₂ grains, andexamined for the presence or absence of SGP-D1 and SGP-B1 by subjectingto electrophoresis so as to select those lacking both SGP-D1 and SGP-B1.The proximal halves corresponding to the selected distal halves areseeded to obtain plants lacking both SGP-D1 and SGP-B1. The obtainedplant is crossed with another wheat cultivar lacking only SGP-A1 (Sgp-A1null) so as to obtain new F₁ seeds. The new F₁ seeds will beheterozygous for all of Sgp-A1, Sgp-B1 and Sgp-D1, and therefor all ofSGP-A1, SGP-B1 and SGP-D1 will be detected in endosperms of the new F₁seeds. When new F₁ plants which have grown from the new F₁ seeds areself-pollinated, new F₂ seeds will segregate with regard to each ofSgp-A1, Sgp-B1, and Sgp-D1 alleles at the probability of one out of four(¼). Thus, from the entire new F₂ seed population, a new F₂ seed beingnull as to all of Sgp-A1, Sgp-B1 and Sgp-D1 is obtained at theprobability of one out of sixty-four ({fraction (1/64)}; i.e., ¼multiplied by ¼, further multiplied by ¼), theoretically.

Starches are purified from distal halves of the obtained new F₂ grains,and examined for the presence or absence of SGP-A1, -B1 and -D1 bysubjecting the SGPs to electrophoresis so as to select those lacking allof SGP-A1, -B1 and -D1. The proximal halves corresponding to theselected distal halves are seeded to obtain plants lacking all ofSGP-A1, -B1 and -D1.

While an exemplary crossing process has been described above, the orderof crossing is not limited to the order described above. Wheat cultivarslacking only SGP-A1 (SGP-A1 null wheat) include Chousen 30, Chousen 57and the like. Wheat cultivars lacking only SGP-B1 (SGP-B1 null wheat)include Kanto 79, and the like. Wheat cultivars lacking only SGP-D1(SGP-D1 null wheat) include Turkey 116, and the like. Seed for wheatlines Turkey 116 (FERM BP-08426). Kanto 79 (FERM BP-08423). Chousen 30(FERM BP-08424) and Chousen 57 (FERM BP-08425) were deposited on Jul. 9,2003 under the Budapest Treaty with the International Patent OrganismDepository. National Institute of Advanced Industrial Science andTechnology located at AIST Tsukuba Central 6, 1-1, Higashi 1-ChomeTsukuba-shi, Ibaraki-ken 305-8566 Japan. Other such cultivars may beobtained by screening according to the method described in Yamamori andEndo, supra.

Tetraploid wheat, e.g., durum wheat, which lacks SGP-1 can also beobtained in a manner similar to that for SGP-1 null hexaploid wheat. Forexample, SGP-1 null tetraploid wheat may be produced by first obtaininghexaploid wheat (2n=42) lacking both SGP-A1 and -B1, crossing theobtained hexaploid wheat with durum wheat, and then selecting progeniesbeing tetraploid (2n=28) and lacking both SGP-A1 and -B1. Alternatively,SGP-1 null tetraploid wheat may be produced by first crossing two durumwheat cultivars which lack SGP-A1 and -B1, respectively,self-pollinating the obtained cross, and then selecting progenies whichlack both SGP-A1 and -B1.

SGP-1 null hexaploid wheat may alternatively be produced by crossingtetraploid wheat lacking both SGP-A1 and -B1 with Aegilops squarrosahaving genome organization of DD and lacking SGP-D1 so as to obtaintriploid individuals, subjecting the obtained triploid individuals to adoubling of chromosomes such as a colchicine treatment so as to obtainhexaploid progeny, and then obtaining hexaploid progenies which lack allof SGP-A1, SGP-B1 and SGP-D1.

The present invention has been made based on a discovery that wheatlacking SGP-1 produces novel starch having a high level of apparentamylose content which, to the extent that the present inventor is awareof, has not been previously known in the art. Wheat starch and wheatflour of the present invention may be obtained by any method with whichwheat lacking SGP-1 can be produced, and such method is not limited tothe cross breeding as described above. For example, wheat lacking SGP-1may alternatively be obtained by first treating wheat having SGP-1 witha mutagen, and then screening the treated wheat plant or progeny of thetreated wheat plant obtained by self-pollinating the treated wheatplant, for the absence of all of SGP-A1, SGP-B1 and SGP-D1.Alternatively, when a wheat plant lacking one or two of SGP-A1, SGP-B1and SGP-D1 is found in a wheat plant population obtained by e mutagenictreatment, crossing process(es) may further be performed using suchwheat plant as a parental plant so as to obtain wheat plant lackingSGP-1.

The mutagen may be any appropriate mutagen including a physical mutagensuch as ionizing radiation and a chemical mutagen. The physical mutagensinclude gamma ray, X ray, fast neutron, thermal neutron, beta ray, andthe like. The chemical mutagens include ethyl methanesulfonate (EMS),N-methyl-N-nitrosourea (MNU), diethyl sulfate (dES), sodium azide(NaN₃), and the like. Appropriate methods for treating wheat plant withsuch mutagens, and appropriate conditions including what kind of wheatmaterial is to be used with such a treatment are known to, and will beselected by, those skilled in the art.

Moreover, wheat lacking SGP-1 may alternatively be produced by anyappropriate genetic engineering approach known in the art, includingprotoplast fusion, homologous recombination, antisense technique, andthe like. Those skilled in the art can appropriately select one of theseand other approaches, and combinations thereof.

3. Production of Wheat Starch and Wheat Flour with High Amylose Content

Wheat starch having the high amylose content of the present inventionmay be prepared by isolating starch from wheat seed lacking SGP-1according to any appropriate method known in the art. Wheat flour havingthe high amylose content of the present Invention may be prepared bymilling the wheat seed lacking SGP-1 according to any appropriate methodknown in the art.

Wheat starch and wheat flour of the present invention are believed to benovel materials characterized by having a high level of apparent amylosecontent which has not been previously known in the art. Such wheatstarch and wheat flour may be useful in various industrial and foodapplications. Moreover, wheat starch of the present invention may alsobe useful for the purpose of researching the correlation between thestructure of glucose polymer and starch properties. Furthermore,modified wheat of the present invention may be useful as a breedingmaterial for developing wheat which produces starch having an amylosecontent as high as that of maize (60%-70%).

EXAMPLES Example 1 Production of Wheat Lacking SGP-1 (SGP-1 Null Wheat)

1. Plant Material

To produce a wheat which lacks SGP-1 (SGP-1 null wheat), the followingfour parental wheat (Triticum aestivum L.) cultivars were used: Chousen30 (C 30) and 57 (C 57) lacking SGP-A1; Kanto 79 (K 79) lacking SGP-B1and Turkey 116 (T 116) lacking SGP-D1 (see Table 1).

First, T 116 and K 79 were crossed to obtain F₁ seeds. F₁ plants whichgrew from the F₁ seeds were self-pollinated to obtain F₂ seeds. Starcheswere purified from the distal half of the F₂ seeds. SDS-polyacrylamidegel electrophoresis (SDS-PAGE) was performed using the purified starchesso as to examine the presence or absence of SGP-D1 and -B1. As a result,F₂ seeds lacking both SGP-D1 and -B1 from cross K 79/T 116 wereselected. Purification of the starches and SDS-PAGE will be described ingreater detail below.

F₂ plants which grew from the selected F₂ seeds lacking both SGP-D1 and-B1 were pollinated by either of C 30 and C 57, both lacking SGP-A1, toobtain new F₁ seeds. New F₁ plants grown from the new F₁ seeds wereself-pollinated to obtain new F₂ seeds. Starches were purified from thedistal half of the new F₂ seeds. SDS-polyacrylamide gel electrophoresis(SDS-PAGE) was performed using the purified starches so as to examinethe presence or the absence of SGP-A1, -B1 and -D1. As a result, fromthe cross (K 79/T 116)F₂//C 30 or C 57, variant progeny (new F₂ plant)lacking SGP-1 was selected.

TABLE 1 Alleles for Sgo-1 in wheat materials used to produce wheat withno SGP-1 (SGP-1 null wheat) Sgp-1 Wheat −A1 −B1 −D1 SGP-1 null b b bTurkey 116 a a b Kanto 79 a b a Chousen 57 b a a Chouscn 30 b a aChinese Spring a a a Norin 61 a a aIn Sgp-1 alleles, a indicates standard allele in cv-Chinese Spring whichproduces the protein coded by the gene, while b indicates null allelewhich does not produce the coding protein. The allele -A1 belongs to Agenome, -B1 to B genome and to -D1 to D genome. Four wheats (Turkey116-Chousen 30) were used to produce SGP-1 null wheat. Two cultivars(Chinese Spring and Norin 61) were controls for analyzing starchproperties.2. Starch and Starch Granule Preparation

Starches from the distal half of F₂ seeds for screening were preparedaccording to Sulaiman and Morrison (J. Cereal Sci. 12:53-61 (1990)),using 80% CsCl.

For characterization of starch, starch granules were prepared accordingto Echt and Schwartz, supra. Hammer-crushed wheat seeds were homogenizedin a protein extraction buffer (55 mM Tris/HCl, pH 6.8, 2.3% SDS, 5%2-mercaptoethanol and 10% glycerol). This suspension was passed througha 50 μm nylon mesh to remove large seed coats. After centrifugation at13,500 rpm for 2 min, a yellowish layer on white starch pellet wasremoved by spatula, and the remaining white starch pellet was suspendedin the extraction buffer. This procedure was repeated twice, then thestarch was washed twice by distilled water and twice by acetone and airdried.

3. SDS-polyacrylamide Gel Electrophoresis

SDS-polyacrylamide gel electrophoresis of starch granule proteins (SGPs)was performed as described by Yamamori and Endo, supra. An amount of 5mg of starch prepared from ten (10) mature grains or 5 mg of starch froma distal half of F₂ grain was gelatinized in 70 μl of the proteinextraction buffer by heating for 5 min. After centrifugation for 5 minat 13,500 rpm, the supernatant (15 μl) was subjected to electrophoresis.For the resolution gel, acrylamide in a concentration of 12.5% andBIS-acrylamide in a low concentration (acrylamide/BIS-acrylamideconcentration of 30.0.135) were used. Proteins were visualized by silverstaining (Silver stain kit; Wako Pure Chemical Industries, Ltd. Japan).

For characterization of starch, cultivar Chinese Spring or Norin 61having all of SGP-A1, -B1 and -D1 were used as controls.

4. Results

SDS-PAGE analysis of 968 new F₂ seeds from the cross (Kanto 79/Turkey116)F₂//Chousen 30 or Chousen 57 found that four seeds yielded no SGP-1.New F₂ seeds were classified into eight categories based on Sgp-1alleles or the presence or absence of SGP-A1, -B1 and -D1. Since thethree genes, Sgp-A1, -B1 and -D1 are located on different chromosomes,the expected ratio for the eight categories is 27:9:9:9:3:3:3:1 (seeTable 2). However, the observed number did not fit the expected ratio(χ²=14.26, P<0.05). Seed fertility of F₃ plants derived from new F₂plants (SGP-1 null) was 94%, while fertility of cultivar Chinese Springwas 97%. This shows fertility of the SGP-1 null wheat was normal. TheSGP-1 null wheat used in Examples 2-4 below was obtained from (Kanto79/Turkey 116)F₂//Chousen 57.

TABLE 2 Segregation of F₂ from (Kanto 79,Trukey 116)F₂// Chousen 30 orChousen 57 Alleles of Sgp-1 Number of F₂ Expected χ² -A1 -B1 -D1 seedsobserved ratio value a a a 424 27  0.60 b a a 150 9  1.41 a b a 125 9 0.91 a a b 143 9  0.35 a b b 47 3  0.06 b a b 38 3  1.20 b b a 37 3 1.55 b b b 4 1  8.18 Total 968 64 14.26* Sgp-A1a, -B1a and -D1a arestandard alleles in cv Chinese Spring. Sgp-A1b, -B1b and -D1b are nullalleles lacking each SGP-1. *significant difference from the expectedratio at the 5% level by χ² test.

Example 2 Decrease in Other Starch Granule-bound Proteins In SGP-1 NullWheat

In addition to SGP-1, wheat starch granules carry three granule-boundproteins, i.e., waxy protein, SGP-2 and SGP-3. In new F₂, F₃ and F₄seeds of the SGP-1 null wheat, SGP-2 and -3 decreased considerably whilethe waxy protein did not, as observed on a gel of SDS-PAGE. The resultfor the F₄ seeds is seen in lane 5 of FIG. 1. To examine how much theSGP-2 and -3 decreased in the SGP-1 null wheat, 1, ½, ¼, ⅛, {fraction(1/16)} and {fraction (1/32)} sample volumes of the cultivar ChineseSpring were subjected to electrophoresis and the thickness of SGP-2 and-3 bands detected by silver staining were compared to one volume fromthe SGP-1 null wheat. As a result, it was found that the elimination ofSGP-1 was accompanied with a decrease of both SGP-2 and -3 to about{fraction (1/16)} as compared with the cultivar Chinese Spring.

Example 3 Measurement of Blue Value and λ_(max) of Wheat Lacking SGP-1(SGP-1 Null Wheat)

To characterize starch components, the present inventor measured theblue value (absorbance at 680 nm) and maximum absorbance (λ_(max)) ofiodine-starch complex from the SGP-1 null wheat (F₄ seeds), its parentsand cultivar Chinese Spring (see Table 3). Higher blue value indicatesthat the apparent amylose content of the SGP-1 null wheat was higherthan those of the others.

The absorbance at 680 nm (blue value) and maximum. absorbance (λ_(max))of the iodine-starch complex were determined according to Konishi et al.(Agric Biol. Chem. 49:1965-1971, 1985). An amount of 10 mg of starch wasgelatinized in 1 ml of 1N NaOH for one hour at 40° C., and neutralizedby 9 ml of {fraction (1/9)} M acetic acid. Then, 1 mg of gelatinized andneutralized starch was mixed with 2 mg of I₂ and 20 mg KI, and distilledwater was added to make a 25 ml solution. Absorption curves ofstarch-iodine complexes were measured at 500-700 nm, and blue value andλ_(max) were recorded.

Example 4 Measurement of Amylose Content of SGP-1 Null Wheat

To confirm that wheat lacking SGP-1 has a high apparent amylose content,the amylose content was measured by calorimetric method and amperometrictitration as follows.

(1) Colorimetric measurement based on Iodine coloration was performedfollowing the method of Kuroda et al. (Jpn. J. Breed. 39 (Suppl.2):142-143, 1989) using an auto-analyzer (Bran Lubbe. Co.). An amount of35 mg of starch was gelatinized in 5 ml of 0.75 N NaOH and 25% aqueousethanol, and neutralized by acetic acid. Absorbance at 600 nm of starchiodine complex was measured by colorimeter. For control, two wheatstarches were used. A first control, wheat starch purchased from WakoPure Chemicals Ltd. (Japan) contained 31.2% amylose as determined by theauto-analyzer using potato amylose and amylopectin as standards, and asecond control, waxy wheat starch contained 0.6% amylose.

The amylose content of the starch from the SGP-1 null wheat was as highas 37.3% (see Table 3). In contrast, Norin 61 and Chinese Spring had anamylose content of 28.2% and 29.6%, respectively. Thus, the amylosecontent of the SGP-1 null wheat starch was higher than those ofcultivars Norin 61 and Chinese Spring by about 8% to 9%. The three wheatcultivars used as crossing parents, i.e., Turkey 116, Kanto 79 andChousen 57, had amylose contents ranging from 23.9% to 30.3%.

(2) Amperometric titration (Fukuba and Kainuma, “Quantification ofamylose and amylopectin” in Starch Science Handbook (Nakamura M. andSuzuki S., eds) Tokyo: Asakura Shoten, pp 174-179, 1977) was performedusing defatted starch with an iodine amperometric titration device(Model 3-05, Mitamura Riken Kogyo, Japan). Amylose content of the starchwas calculated by assuming that 20 mg of iodine can bind to 100 mg ofpure wheat amylose. The starch concentration of the solution used wasdetermined by the phenol-sulfuric acid method (Dubois et al., Anal.Chem. 28:350-356, 1956) with glucose as a standard.

The amylose content of the starch from the SGP-1 null wheat was 37.3%(see Table 3). In contrast, Norin 61 and Chinese Spring had an amylosecontent of 26.6% and 29.3%, respectively. Thus, the amylose content ofthe SGP-1 null starch was higher than that of cultivars with SGP-1,Norin 61 and Chinese Spring by about 8% to 11%. The three wheatcultivars used as crossing parents had amylose contents ranging from23.54 to 29.8%.

TABLE 3 Maximum absorbance (λmax), absorbance at 680 nm (blue value) ofstarch-iodine complex and amylose content of wheats (F₄ seeds) λmax BlueAmylose content (%) Wheat (nm) value Colorimetric Titration SGP-1 null(F₄) 602 ± 6 0.485 ± 0.023 37.3 ± 0.8 37.3 ± 0.8 Turkey 116 589 ± 50.370 ± 0.011 30.3 ± 0.2 29.8 ± 0.5 Kaoto 79 565 ± 0 0.307 ± 0.004 23.9± 0.5 23.5 ± 0.1 Chousen 57 591 ± 1 0.365 ± 0.003 29.4 ± 0.1 28.1 ± 0.2Chinese Spring 586 ± 3 0.358 ± 0.013 29.6 ± 0.1 29.3 ± 0.3 Norin 61 —¹⁾— 28.2 ± 0.1 26.6 ± 0.2 ¹⁾These were not examined.Values are means±SD from three replicates for two controls and threeparental wheats. For SGP-1 null, values from eight (titration), ten(calorimetric) and 15 (λ_(max) and blue value) replicates wereindicated.

The above-mentioned measurement results all indicate that the apparentamylose content of the SGP-1 null wheat starch is considerably higherthan that of normal wheat starch.

Various other modifications will be apparent to and can be readily madeby those skilled in the art without departing from the scope and spiritof this invention. Accordingly, it is not intended that the scope of theclaims appended hereto be limited to the description as set forthherein, but rather that the claims be broadly construed.

1. Wheat which is modified to lack SGP-1, comprising wheat starch whichhas an apparent amylose content of about 35% or more.
 2. The wheat ofclaim 1, wherein the apparent amylose content is from about 37% to about40%.
 3. The wheat of claim 1, which is a hexaploid wheat which lacksSGP-A1, SGP-B1 and SGP-D1.
 4. The wheat of claim 2, wherein thehexaploid wheat is obtained by crossing a first wheat lacking a firstprotein selected from the group consisting of SGP-A1, SGP-B1 and SGP-D1,with a second wheat lacking a second protein which differs from thefirst protein and is selected from the group consisting of SGP-A1,SGP-B1 and SGP-D1, to obtain a first F1 cross; self pollinating theresulting first F1 cross to obtain a first F2 cross and selecting afirst null cross lacking said first and second protein from said firstF2 cross; followed by further crossing the said first null cross of thefirst wheat and the second wheat with a third wheat lacking a thirdprotein which differs from the first and second proteins and is selectedfrom the group consisting of SGP-A1, SGP-B1 and SGP-D1 to obtain asecond F1 cross; and self-pollinating the resulting second F1 cross toobtain a second F2 cross and selecting a second null cross lacking saidfirst, second and third protein from said second F2 cross.
 5. The wheatof claim 3, wherein the hexaploid wheat is obtained by crossing a firstwheat lacking a first protein selected from the group consisting of (i)Chousen 30 or Chousen 57, (ii) Turkey 116, and (iii) Kanto 79, with asecond wheat lacking a second protein which differs from the firstprotein and is selected from the group consisting of (i), (ii), and(iii), to obtain a first F1 cross; self pollinating the resulting firstF1 cross to obtain a first F2 cross and selecting a first null crosslacking said first and second protein from said first F2 cross; followedby further crossing the said first null cross of the first wheat and thesecond wheat with a third wheat lacking a third protein which differsfrom the first and second proteins and is selected from the groupconsisting of (i), (ii), and (iii) to obtain a second F1 cross; andself-pollinating the resulting second F1 cross to obtain a second F2cross and selecting a second null cross lacking said first, second andthird protein from said second F2 cross.